Affording to Current Understanding of Molecular Mechanisms of Diseases, Reagents that Modulate Immune Responses are in Great Demand.
The body's defense system against microbes and other chronic diseases is mediated by the two main components of the immune system: the innate immune system and adaptive immune system. Recent advances in the study of the molecular and cellular mechanism of various diseases indicated that both innate and adaptive immune systems are targeted for the prevention and cure of various types of diseases. Innate immunity involves mechanisms that recognize structures, which are characteristic to microbial pathogens but are not present on mammalian cells. Examples of such structures include bacterial liposaccharides (LPS), viral double stranded DNA, and unmethylated CpG DNA nucleotides. The effector cells of the innate immune response system comprise neutrophils, macrophages, and natural killer cells (NK cells). In adaptive immunity, the body's immunological defense systems are stimulated by exposure to infectious agents and these responses increase in magnitude and effectiveness with each successive exposure to that particular antigen. There are two types of adaptive immune responses: (i) humoral immunity, which involves the production of pathogen-specific antibodies by B lymphocytes (B cells), and (ii) cell-mediated immunity, which is regulated by T lymphocytes (T cells). Immune effector cells in the innate and adaptive phases of immune responses can be directly or indirectly involved in the cause of some diseases, and are thus potentially important targets for therapeutics against these diseases. Recently, there has been an increasing number of vaccine strategies used against a variety of disease conditions. Infectious diseases caused by viruses, bacteria and parasites [Targett and Greenwood Malar J. 7 Suppl 1:S10 (2008); Okwor and Uzonna Hum Vaccin. May 12; 5 (2009)] are areas of on-going research using vaccines. Unexpectedly, vaccine strategies may also be effective against diseases such as Alzheimer's and prion diseases, in which the scavenger functions of immune response cells against pathogenic metabolic deposits is ineffective [Wisniewski and Konietzko, Lancet Neurol. 7:805 (2008); Sakaguchi, Protein Pept Lett., 16:260 (2009)]. Autoimmune diseases are also an area in which immune responses against the host self-components could potentially be inhibited at pathogenic level by using vaccine strategies to specific self-reactive T cells and B cells. As the list of diseases that are potential targets for treatment by immunological approaches increases, modulators for the immune competent cells are in greater demand. Improved methods for increasing or repressing immune responses, while following safe guidelines for use in humans, represent a major unmet demand in modern medicine.
B Cell- and APC-Targeting Immunotherapies
The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is broadly expressed by immune, hematopoietic, vascular, epithelial, and other cells, including a wide range of tumor cells. CD40 itself lacks intrinsic kinase or other signal transduction activity, but rather mediates its diverse effects via an intricate series of downstream adapter molecules that differentially alter gene expression depending on cell type and microenvironment. As a potential target for novel cancer therapy, CD40 may mediate tumor regression through both an indirect effect of immune activation and a direct cytotoxic effect on the CD40-expressing tumor.
CD40 is best known as a critical regulator of cellular and humoral immunity via its expression on B lymphocytes, dendritic cells, and monocytes [Grewal and Flavell, Annu Rev Immunol., 16:111 (1998); van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000)].
CD40 is also expressed on the cell surface of many other non-immune cells, including endothelial cells, fibroblasts, hematopoietic progenitors, platelets and basal epithelial cells [Grewal and Flavell, Annu Rev Immunol., 16:111 (1998); van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Young et al., Immunol Today, 9:502(1998); Quezada et al., Annu Rev Immunol., 22:307 (2004)]. The CD40 ligand (CD40L), also known as CD154, is the chief ligand described for CD40 and is expressed primarily by activated T lymphocytes and platelets [van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Armitage et al., Nature, 357:80 (1992)]. Atherosclerosis, graft rejection, coagulation, infection control, and autoimmunity are all regulated by CD40-CD40L interactions [Grewal and Flavell, Annu Rev Immunol., 16:111 (1998); van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000)]. Many tumor cells also express CD40, including nearly 100% of B-cell malignancies and up to 70% of solid tumors.
Physiologically, CD40-induced signal transduction represents a major component of a process known as T-cell “help.” Ligation of CD40 on dendritic cells, for example, induces cellular maturation and activation as manifested by increased surface expression of co-stimulatory and MHC molecules, production of proinflammatory cytokines such as interleukin 12, and enhanced T-cell activation [van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Quezada et al., Annu Rev Immunol., 22:307 (2004)]. CD40 ligation of resting B cells also increases antigen-presenting function and, in addition, induces proliferation and immunoglobulin class switching [van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Quezada et al., Annu Rev Immunol., 22:307 (2004)]. Patients with germ line mutations in either CD40 or CD40L are markedly immunosuppressed, susceptible to opportunistic infections, and have deficient T-cell-dependent immune reactions, including IgG production, germinal center formation, and memory B-cell induction [Allen et al., Science, 259:990 (1993); Ferrari et al., Proc Natl Acad Sci USA, 98:12614 (2001); Etzioni A, Ochs H D. Pediatr Res., 56:519 (2004)]. Similar immunophenotypes are observed in mice deficient in CD40 or CD40L [Castigli et al., Proc Natl Acad Sci USA, 91:12135 (1994); Kawabe et al., Immunity, 1:167 (1994); Renshaw et al., J Exp Med., 180:1889 (1994); Xu et al., Immunity, 1:423 (1994)]. Agonistic CD40 antibodies have been shown to mimic the signal of CD40L and substitute for the function of CD4+ T lymphocytes in murine models of T-cell-mediated immunity [Bennett et al., Nature, 393:478 (1998); Ridge et al., Nature, 393:474 (1998); Schoenberger et al., Nature, 393:480 (1998)]. A key mechanism of this effect is thought to be CD40/CD40L-mediated activation of host dendritic cells. Growing evidence shows that stimulating APC with soluble CD40L or an agonistic anti-CD40 antibody can, at least in part, replace the need for T helper cells and generate antigen presenting cells (APCs) that are capable of priming cytotoxic T lymphocytes (CTL). To develop pharmacotherapeutic reagents targeting the CD40/CD40L pathway, series of soluble CD40L fusion proteins were disclosed. In one invention, CD40L was joined to antigens to deliver CD40-costimulation signal and antigens together to B cells and APCs (WO/2003/063899). In another invention, the conjugate of CD40L and a Toll-like receptor ligand, Flagellin, was created to trigger a synergistic activation signaling between CD40 and TLR5 in B cells and APCs (WO/2007/103048). All these innovations aimed to use CD40L fusion proteins as vaccines by stimulating antigen-specific B cell and APCs in vivo.
T Cell Targeting Immunotherapies
Two types of major T lymphocytes have been described, CD8+ cytotoxic lymphocytes (CTLs) and CD4+ helper cells (Th cells). CD8+ T cells are effector cells that, via the T cell receptor (TCR), recognize foreign antigens presented by class I major histocompatibility complex (MEW) molecules on, for instance, virally or bacterially infected cells. T helper cells are involved in both humoral and cell-mediated forms of effector immune responses. With respect to the humoral or antibody immune response, antibodies are produced by B lymphocytes through interactions with Th cells. Specifically, extracellular antigens, such as circulating microbes, are taken up by specialized APCs, processed, and presented in association with class II MHC molecules to CD4+ Th cells. These Th cells in turn activate B lymphocytes, resulting in antibody production. In contrast, the cell-mediated immune response functions to neutralize microbes that inhabit intracellular locations after infection of a target cell.
According to the two-signal model, optimal activation of antigen-specific T lymphocytes requires specific antigen recognition by lymphocytes (signal 1′) and additional signals (called ‘signal 2’ or co-stimulatory signals). In the absence of signal 2, lymphocytes fail to respond effectively and are rendered anergic. Signal 1 is provided by the interaction of the peptide-antigen-MHC complex with the TCR. Signal 2 is delivered to T cells by co-stimulatory cell surface molecules expressed on APCs. The process of co-stimulation is of therapeutic interest because the manipulation of co-stimulatory signals might provide a means either to enhance or to terminate immune responses.
The B7-1/B7-2-CD28/CTLA-4 pathway is the best-characterized T-cell co-stimulatory pathway and is crucial in T-cell activation and tolerance [Karandikar et al., J. Neuroimmunol. 89:10 (1998); Oosterwegel et al., Curr. Opin. Immunol. 11:294 (1999); Salomon and Bluestone, Annu. Rev. Immunol. 19:225 (2001); Sansom, Immunology, 101:169 (2000); Chambers et al., Annu. Rev. Immunol., 19:565 (2001)]. The B7-1/B7-2-CD28/CTLA-4 pathway includes two B7 family members, B7-1 (CD80) [Freeman et al., J. Exp. Med. 174:625 (1991); Freedman et al., J. Immunol. 137:3260 (1987); Yokochi et al., J. Immunol. 128:823 (1982)] and B7-2 (CD86) [Freeman et al., Science 262:909 (1993); Freeman, et al., J. Exp. Med. 178:2185 (1993); Azuma, et al. Nature 366:76 (1993)], that have dual specificity for two CD28 family members, the stimulatory receptor CD28 antigen-receptor signaling [Aruffo and Seed, Proc. Natl Acad. Sci. USA 84:8573 (1987); Gross et al., J. Immunol., 144:3201 (1990)], by promoting T-cell survival and thereby enabling cytokines to initiate T-cell clonal expansion and differentiation [Thompsonet et al., Proc. Natl. Acad. Sci. USA 86:1333 (1989); Lucas et al., J. Immunol., 154:5757 (1995); Shahinian et al., Science 261:609 (1993); Sperling et al., J. Immunol., 157:3909 (1996); Boise et al., Immunity 3:87 (1995)]. CD28 also optimizes the responses of previously activated T cells, promoting interleukin 2 (IL-2) production and T-cell survival.
Several members of the tumor necrosis factor receptor (TNFR) family function as co-stimulatory receptors after initial T cell activation. These include CD27, 4-1BB (CD137), OX40 (CD134), HVEM, CD30 and GITR [reviewed in Watts, Annu Rev Immunol., 23:23 (2005)].
To develop immunotherapeutic reagents targeting T cells, soluble co-stimulatory receptor extracellular fragments, soluble ligand extracellular fragments, fusion proteins or agonistic antibodies against receptors or specific ligands have been studied. Alternatively, un-agonistic soluble ligands or un-agonistic antibodies have been used to block co-stimulatory receptor signaling. Either by increasing or reducing the extent of T cell costimulation, the use of ligand-fusion proteins or antibodies has shown pharmaceutical benefits to diseases including autoimmune diseases, proliferative disorders such as cancer, or infectious diseases.
Death Receptor “Fas” and Disease Therapy
Fas (CD95), a member of the tumour-necrosis factor receptor (TNFR) superfamily, was originally described as a lymphocyte receptor that can induce apoptosis [Yonehara et al., J. Exp. Med., 169:1747 (1989); Trauth et al., Science, 245:301 (1989)]. Fas is expressed in many types of tissue including glia cells, neurons and neuronal cell lines [Shinohara et al., Cancer Res., 60:1766 (2000); Gomez et al., J. Neurosci. Res., 63:421 (2001); Becher et al., Neurosciences, 84:627 (1998); Raoul et al., J. Cell Biol. 147:1049 (1999); Raoul et al., Neuron, 35:1067 (2002); Matsushita et al. J. Neurosci. 20:6879 (2000); Cheema et al., J. Neurosci., 19:1754 (1999)]. The interaction between CD95 (Fas) and its ligand (Fas-ligand, or FasL) functions to limit the duration of the immune response and/or life-span of activated lymphocytes. Apoptosis induced by Fas-FasL binding serves to clear activated self-reactive lymphocytes. Problems caused by altering this pathway have been demonstrated in animals with defects in Fas↔Fas-ligand interactions. Mice having mutations, which inactivate Fas or FasL, develop numerous disorders including autoimmune pathology resembling that seen in patients with rheumatoid arthritis or systemic lupus. It has been demonstrated that injection of FasL-expressing virus into the joints of mice with collagen-induced-arthritis, results in apoptosis of synovial cells and relief of arthritis symptoms [Zhang et al., in J. Clin. Invest., 100:1951 (1997)]. Expression of the Fas ligand reduces the number of activated inflammatory cells, which play a role in the pathogenesis of autoimmune disease. Therefore, a gene therapy strategy for introducing FasL into the joints of rheumatoid arthritis patients could function to improve disease pathology by leading to destruction of the infiltrating mononuclear cells.
Soluble Fas ligand and receptor have also been shown to be associated with tissue damage and other adverse effects. Administering an agonistic anti-Fas antibody resulted in organ damage to mice [Galle et al., J. Exp. Med. 182:1223 (1995)]. Mice injected intraperitoneally with the agonistic antibody died within several hours, and analyses revealed that severe liver damage by apoptosis was the most likely cause of death.
Fas engagement by FasL, or by antibodies against Fas, initiates binding of the intracellular death domain of Fas to an adaptor protein, the Fas-associated death domain (FADD), which couples Fas to the caspase cascade. Caspase 8 (also known as FADD-like interleukin-1 converting enzyme; FLICE) is the most upstream caspase in the apoptosis pathway, and its cleavage is a hallmark of Fas-induced death [Nagata, Cell 88: 355 (1997); Medema et al. EMBO J. 16:2794 (1997)]. Fas-mediated death signals can be inhibited by the FLICE inhibitory protein (FLIP), which blocks caspase 8 binding to FADD [Irmler et al. Nature 388:190 (1997)]. The activation of a cascade of successive caspase cleavages finally results in the activation of endonucleases that catalyse DNA breakdown into nucleosome-sized fragments, a characteristic feature of apoptosis [Nagata, Cell 88:355 (1997)].
In addition to apoptosis, Fas has been reported to mediate diverse proliferative and regenerative functions, including co-stimulatory signalling during T-cell activation [Alderson et al. J. Exp. Med., 178:2231 (1993); Alderson et al. Int. Immunol. 6:1799 (1994); Desbarats et al. Proc. Natl. Acad. Sci. USA 96:8104 (1999)], induction of angiogenesis [Biancone et al. J. Exp. Med. 186:147 (1997)], and liver regeneration after partial hepatectomy [Desbarats and Newell, Nature Med. 6:920 (2000)].
In contrast to the well-characterized apoptotic pathway, relatively little is known about the signalling pathways involved in Fas-mediated growth induction, although Fas has been shown to activate the extracellular-signal regulated kinase (ERK) pathway [Trauth et al., Science 245:301 (1989)]. ERK, a serine/threonine kinase activated by mitogen-activated protein kinase (MAPK)/ERK kinase (MEK1), mediates the cellular response to many different growth and differentiation factors [reviewed in Fukunaga and Miyamoto, Mol. Neurobiol., 16:79 (1998)]. Notably, activation of ERK prevents Fas-induced apoptosis and, conversely, inhibition of ERK prevents Fas-induced proliferation, suggesting that the MEK1/ERK pathway is involved in the transduction of Fas-mediated growth signals [Trauth et al., Science 245:301 (1989); Holmstrom et al., EMBO J., 19:5418 (2000); Kataoka, et al. Curr. Biol., 10:640 (2000)]. At present, it is not clear how Fas engagement in T-cell co-stimulation, and in the regeneration of liver and nerves, bypasses an apoptotic signal and promotes a regenerative or co-stimulation signal.
Soluble Fas-ligands has been useful reagents to induce pathological cell-specific cell death. For example, a fusion protein that connected interleukin-2-IgFc-FasL was used to kill auto-reactive T cells in autoimmune disease therapy [Bulfone-Paus et al., Transplantation. 69:1386 (2000)]. In a similar approach, the fusion protein of CD40 extracellular domain and FasL extracellular domain, CD40-FasL, showed that cell death is contingent on the binding of CD40 to CD40L expressed on target cells [Siegmund et al., J Mol Med. 84:785(2006)]. A fusion construct comprised of VEGF and FasL was found to effectively kill cancer cells by a synergistic effect between VEGF signaling and Fas signaling (WO/2007/022273). Additionally, a fusion protein containing a DC20-specific antibody fragment and soluble FasL, ScFvRit:sFasL [Bremer et al., Cancer Res., 68:597 (2008)], was applied to non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia. This fusion protein efficiently activated CD20 signaling and Fas cell death signaling, resulting in a far superior proapoptotic activity, compared with co-treatment with anti-CD20 antibody (rituximab) and soluble FasL. Therefore Fas ligand-based fusion proteins have shown promising results in the field of autoimmune diseases and cancer therapy by stimulating Fas-induced death signaling in pathological cells. Fas-associated regenerative or co-stimulation signaling has not been exploited for target cell specific therapy.
Immune Suppressive Molecules
Traditionally, various steroids and inhibitors that block the cell activation and growth signaling, such as FK506 or Rapamycin, are broadly used. Some drawbacks to the use of these reagents are that they are not specific to lymphocytes and their use is often accompanied by serious side effects. The first therapeutic agents (immune suppressors) were mostly non-specific and inhibited cellular proliferation [Van Assche et al., Curr Opin Gastroenterol. May 5 (2009); Arias et al., Transplant Rev (Orlando) 23:94 (2009); Ng et al., Front Biosci., 14:1627 (2009)]. These treatments generally led to serious side effects due to intrinsic lack of pharmacospecificity. Later, cyclosporin A (CsA) was the first of a new generation of immunosuppressants with a ‘site-specific’ mode of action. Mechanistically, CsA mediates its in vivo effects by repressing lymphocyte activation at an early stage. Due to a low degree of myelotoxicity, CsA was considered as an attractive therapeutic drug in clinical transplantation for inhibiting lymphocytic activities without affecting either phagocytosis or migration of the reticulo-endothelial system. In 1978, CsA was tested clinically and due to its strong efficacy was used worldwide in a majority of the transplant centers to maintain graft survival post surgery [Goumenos, Expert Opin Pharmacother. 9:1695 (2008); Beauchesne, Drug Dev Ind Pharm. 33:211 (2007)]. In the meantime, much work has been put into the design of new therapeutic strategies that would present lower side effects but retain substantial efficacy. Based on research on the antigen specific T cell activation by TCR and in the recent application of the non-stimulatory CD3-specific humanized antibody (Alegre et al., J. Immunol., 155:1544 (1995), blockade of co-stimulatory receptors (i.e. CD28) with CTLA4-Ig and CD40 with anti-CD40L antibody has been attempted. Human-specific humanized non-activating anti-CD3 antibody (teplizumab) was FDA approved to prevent the T cell response that causes T cell immune deficiencies in human.
Thus far, inhibition of co-stimulatory receptors or T cell receptors (by non-activating anti-CD3 antibody) has been partially effective in inducing antigen-specific immunological tolerance.
APCs and T cells, or between T cells and B cells are tightly regulated by cell surface receptors and their counter-receptors (ligands). Therefore various techniques and reagents to facilitate or repress major receptor interactions and their signaling mechanisms have been developed for disease therapeutic purposes. Receptor agonistic or blocking antibodies, soluble extracellular domain of ligand fusion proteins, soluble death receptor ligand fusion proteins and mitogenic or immunosuppressive substances of bacterial or plant origin as described supra showed levels of efficacy beneficial to the disease therapy, although the toxic side effects are often accompanied by strong pharmacotherapeutic efficacy.
The present invention provides methods to develop powerful target cell-specific immune-stimulating fusion proteins, which could lead to the effective immunotherapy of various diseases.